J.J.P. Baars, A.S.M. Sonnenberg and L.J.L.D. Van Griensven. Mushroom Experimental Station, Horst 5960 AA, The Netherlands.
Pulsed field electrophoresis (CHEF) of intact chromosomes of the white button mushroom Agaricus bisporus revealed that chromosome IX can vary considerably in length. Length variations were observed during vegetative propagation in both parental nuclei of strain Horstreg. Ul. Hybridisation of CHEF-blots with a genomic clone containing all the rRNA genes showed that these are located on chromosome IX. Sall restriction, which leaves the rDNA unit intact, and a subsequent digestion of the Sall fragment with BamHl, which cuts once in the rDNA unit, revealed that the rDNA units are organized as a head-to-tall cluster on chromosome IX. The length variation of chromosome IX was exclusively due to variation of the number of rDNA repeats in the cluster, i.e. when chromosome IX specific probes were used located outside the rDNA cluster, no variations in length were found.
The length of the rDNA cluster in one parental line varied between 220 to 360 kb and in the other between 430 and 860 kb. Assuming the length of the rDNA unit 9.2 kb, the copy number of rDNA repeats in the parental lines varied between 24 and 34 and between 47 and 93, respectively.
Often, more than two chromosomal bands were observed in fruit bodies and in vegetative clones of heterokaryons. Occasionally, a ladder of bands was found when Sal I digested chromosomal DNA was separated by CHEF and hybridized with the rDNA probe indicating the hypervariability of the number rDNA repeats in A. bisporus. Currently, influences of culture conditions are investigated on the number of rDNA repeats and the effect of this variation on strain behaviour.
Inheritance of Resistance to Streptomycin, Blasticidin and Dimethomorph in Phytophthora infestans
Bagirova S.F., An Jian Li, Dyakov Y.T. Department of Mycology and Algology, Moscow State University, Moscow 119899, Russia.
Induced streptomycin- (Str,900 mkg/ml) and blasticidin- (Bl, 120 mkg/ml) resistant mutants were crossed on oatmeal agar. Fl progeny of this pair was found to be resistant to Str and Bl in concentration 900 mkg/ml and 60 mkg/ml, respectively. Dominant to Str and semi-dominant to Bl inheritance agree with Poedinok and Dyakov (1981) reported resistance at somatic recombination of drug-mutants. Induced dimethomorph-resistant ( Dm, 8 mkg/ml ) mutant accumulated two step-mutations was crossed with compatible susceptible to Dm strain ( lethal dosage 2 mkg/ml ). Both of mutations occurring in diploid fungus are suggested to be dominant. 80 isolates of progeny were established with susceptibility and 2 with resistance to 8 and 6 mkg/ml. Segregation appears to be different from Mendelian inheritance pattern. Lethality of Dm-resistant zygotes and epistatic effect were assumed. Negative effect of Dm-resistant mutations on fitness of isolates was found.
The Relationship Between Conversion at the AM Locus in Neurospora crassa and Crossing over
Frederick J Bowring and David E.A. Catcheside. School of Biological Sciences, Flinders University, SA 5042, Australia.
The incidence of crossing over between flanking markers during meiosis is enhanced when gene conversion is observed at an intervening locus. This is taken as evidence that gene conversion and crossing over are intimately associated. Estimates of the level of association between conversion and crossing over in Neurospora based on closely flanking genetic markers are similar to that for yeast ( r ~ 0.33) (Perkins et al 1993). The Neurospora am locus is not extraordinary in this respect ( r = 0.26).
We have used restriction site and sequence polymorphisms at the am locus to re-examine recombination events in Neurospora at a resolution higher than is possible using conventional genetic flanking markers. We find a much lower association of conversion and crossing over (r = 0.07). Only 7% of am conversions enjoy a crossover nearby.
The modal minimum conversion tract length is about 741 base pairs, similar to that observed in yeast. In 8 of 14 strains which had a crossover at am, this was remote from the conversion event. In the remaining 6 strains, no evidence of gene conversion was detected. The spectrum of recombination events at am is similar to that seen at a number of loci in yeast, suggesting that am is not atypical, adding weight to the argument that the level of association between conversion and crossing over may have been generally overestimated. This suggests either that the two modes of recombination result from distinct mechanisms or that there is a mechanism that strongly biases resolution of a common intermediate, such as a Holiday junction, in favour of preserving the parental combination of flanking markers.
Perkins, D. D. et al, 1993 Genetics, 133: 690-691.
Molecular Analysis of Meiotic Reconmination in the Histidine-3 Region of Neurospora
P. Jane Yeadon and David E. A. Catcheside. School of Biological Sciences, Flinders University, SA 5001, Australia
Meiotic recombination in Neurospora crassa is differentially regulated by at least three trans acting genes: rec-1, rec-2 and rec-3 each polymorphic in the wild collections used to establish the commonly used laboratory strains (reviewed by Catcheside 1986). In each case dominant alleles (rec) are known to reduce recombination in specific target regions on at least two chromosomes. Collectively, the three rec loci influence recombination in approximately one third of the genome sampled and cause about ten fold variation in recombination at their target sites. Recombination in the his-3 region is influenced both by rec-2, which affects crossing over between his-3 and ad-3 and recombination across the his-3 gene, and by the cis acting element cog, which is also polymorphic amongst laboratory strains. The dominant allele cog+ permits higher recombination but events initiated by either allele are blocked by rec-2+. It is presumed that cog like recombinators with the relevant rec gene sensitivity exist within all DNA segments under rec control.
We have found that cog is within highly polymorphic DNA and have used the polytnorphisms to map cog to a region 2.3 - 3.2 kb 3' of his-3 (Yeadon and Catcheside 1995). We have also found substantial polymorphism within the his-3 coding region, providing markers for determining the position of recombination events. We report here that although events stimulated by cog which lead to recombination between his-3 alleles only rarely have termini within cog, there are regons where termination is common; that conversion tracts are discontinuous in some recombinants; and that the chromosome cog+ is preferentially the recipient of information unless rec-2 + is present.
Catcheside DEA (1 986) Genet Res Camb 47:157-165 Yeadon PJ & Catcheside DEA (1995) Current Genetics 28: 155-163.
Isolation and characterisation of the gene encoding isocitrate lyase in Coprinus cinereus
P.T. Chaure1, L.A. Casselton1 and I. F. Connerton2. 1 Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK and 2 Institute of Food Research, BBSRC, Earley gate, Reading, RG6 2EF, UK.
Like many other microorganisms, the basidiomycete fungus Coprinus cinereus can utilize acetate as sole carbon source for growth. Acetate is utilized via acetyl CoA through the action of the mobilising enzyme acetyl CoA synthetase. Acetyl CoA is metabolised via the anaplerotic glyoxylate pathway which replenishes the TCA cycle in the absence of glycolysis. The two key reactions of this cycle are catalysed by the enzymes isocitrate lyase and malate synthase which bypass the two decarboxylation steps of the TCA cycle.
The structural gene for isocitrate lyase, acu-7, was isolated from a genomic library by heterologous hybridization using a probe derived from the Aspergillus nidulans gene, acuD (Mellon et al. 1987). This gene, together with corresponding cDNA clones has been sequenced and the gene sequence and predicted protein sequence will be presented. The coding sequence is interrupted by three introns, two of which occupy conserved positions with respect to introns in the corresponding genes of A. nidulans and Neurospora crassa. The C. cinereus protein, unlike the protein from these other fungi, has a C-terminal SKL tail which may play a role in import into the glyoxysome.
Progress with promoter analysis will be presented.
Mellon, F.M., Little, PFR. and Casselton, L.A. (1987) Mol. Gen. Genet. 210:352-357.
The Autonomously Replicating Plasmid pAB4-ARpl Transfers Between Nuclei in a Heterokaryon of Aspergillus.
Alfons J.M. Debets. Department of Genetics, Agricultural University, Dreyenlaan 2 6703 HA Wageningen, The Netherlands
Genetic analysis of transformants carrying the autonomously replicating (AR) plasmid pAB4-ARpl were performed in Aspergillus niger and A.nidulans. The following results were obtained. Like in A.nidulans, diploid analyses showed that the AR plasmid segregates independently from chromosomal markers in A.niger. Heterokaryon analyses confirmed that the plasmid is nuclear rather than cytoplasmic in Aspergillus, however upon testing large numbers of conidia, transfer of the AR plasmid from one nuclear genotype to the other in the heterokaryon could be observed. The frequency of this transfer from plasmid bearing to naive nuclei in such heterokaryons is considerably higher for A.niger than for A.nidulans. Parasexual behaviour of these heterokaryons was normal. Upon transfer the plasmid seems to remain unchanged and can be further transferred to other nuclei in heterokaryons. Possible modes of transfer will be considered.
Effect of Medium Composition on Cell Growth and Extracellular Production of Glucose Oxidase by Recombinant Aspergillus niger
H.El-Enshasy, K. Hellmuth and U. Rinas. GBF National Research Center for Biotechnology,-Biochemical Engineering Division, Mascheroder Weg 1, 38124 Braunschweig, Germany
The effect of medium composition on cell growth and extracellular production of glucose oxidase (GOD) by recombinant A. niger NRRL-3 (GOD3-18) was investigated. The recombinant strain carries multiple copies of the god gene with the -amylase-signal peptide from A. oryzae under the control of the gpd-promoter of A. nidulans.
The addition of yeast extract to a mineral salt medium containing glucose as carbon source enhanced cell growth and production of extracellular GOD. However, an increase of the yeast extract concentration above a critical value caused declining extracellular concentrations of GOD.
Studies on the influence of different carbon and nitrogen sources on cell growth and extracellular production of GOD revealed highest productivities using glucose, xylose or mannose as carbon and nitrate as nitrogen source. Again, an increase of the nitrate concentration above a critical value caused a decline in the extracellular production of GOD.
Breeding Systems in the Genus Agaricus
Calvo-Bado, L., Challen, M.P., Elliott, TJ. Department of Microbial Biotechnology, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, U.K.
The breeding systems of homobasidiomycetes are typically homothallic or heterothallic. In the genus Agaricus, which contains the principal cultivated species A. bisporus, clamp-connections are absent, mating reactions may not be evident and fruiting tests are not easily performed. Determining the breeding systems present in Agaricus spp. is therefore not straightforward. This study aims to use RAPD markers to clarify what breeding systems are present in a range of wild Agaricus species. In a heterothallic species, diversity would be expected amongst single spore progeny: in a truly homothallic species uniformity would be expected.
To date five species have been studied, Agaricus nivescens and A. bitorquis which are demonstrably heterothallic, A. subfloccusus which is believed to be homothallic on the basis of fertility of single spore progeny and two other species whose breeding systems have not been classified. In A. subfloccosus the variation between parental strains and their single spore progeny was 2% based on 55 scorable markers with a size range from 340 to 3170 bp. In A. nivescens and A. bitorquis the percentage score fell between 18.2% (32 markers: 660 - 2230 bp) and 11.1% (15 markers: 990-2210 bp) respectively. Of the two undefined species A. silvicola showed 4.3% variation (32 markers: 440-3620 bp) suggesting it is homothallic whilst A. campestris showed 18.9% variation (22 markers: 440 to 3120 bp). The study is being extended to other species and defined markers are being used to test for heterokaryon formation between single spore progeny. The use of RAPDs is clearly informative in determining the breeding system present in an unstudied species.
Population Genetical Aspects of the Incompatibility System of Higher Basidiomycetes
Gutz, Herbert, and Krafzig, Dirk. Institut fur Genetik, Technische Universitat, D-38092 Braunschweig, Germany
In population genetics the kind of sexual reproduction is an important parameter. For instance, to the population of a bisexual species the Hardy-Weinberg law applies if panmixia exists. The situation is more complex in populations of higher basidiomycetes since many different incompatibility factors (IF) may be present and because some species are tetrapolar. For bipolar as well as tetrapolar species, we devised mathematical models which make possible to calculate the intrinsic changes of the IF frequencies in consecutive generations. Regardless of the original composition, all populations approach an equilibrium in which each IF has the frequency 1/N, where N is the number of rare IFs. Some instructive examples will be presented. During the process of convergence, frequency dependent selection takes place in favor of the rare IFs. If in a bipolar species (N > 2), a deleterious gene (relative fitness < 1) is closely linked to an IF, the extinction of this gene is prevented if > (N - 2) / (N - 1).
Presence of Introns in Nuclear 18s rRNA-genes of Ascomycetal Black Yeasts
Gerhard Haase, Andreas Podbielski, Beate Melzer-Krick, and Lydia Sonntag. Institute of Medical Microbiology, Klinikum RWTH Aachen, D-52057 AACHEN, Germany
Fungi belonging to genera of so called “black yeasts” with ascomyccetal affiliation are of medical interest because of their association to variable severe phaeomyphomycoses in human and animals. The pleomorphism exhibited bv these fungi impedes the identification of such clinical isolates. In order to establish a reliable method for differentiation utilizing a molecular approach we sequenced the nuclear 18S rRNA-genes of 36 well characterized strains of black yeasts so far.
Within the 18S rRNA-genes of the following fifteen species we found introns ranging in size from 377 to 475 bp: Exophiala “bergeri" CBS 353.52, Exophiala “gougerotii" CBS 526.76, Exophiala jeanselmei v. heteromorpha CBS 232.33(3) , Exophiala “mesophila ", Exophiala moniliae CBS 520.76, Nadsoniella nigra CBS 535.94,Nadsoniella nigra hesuelica CBS 546.82, Phaeoannellomyces elegans UTMB 1286, Pullularia prototropha CBS 534.94 (3), Rhinocladiella aquaspersa CBS 313.73 (3), Exophiala dermatitidis CBS 207.34 (2), and four allied synanamorphs: Exophiala “ schawii” CBS 292.49 (2), Wangiella dermatitidis ATCC 34 100 (2), “Phaeotheka" dermatitidis KU A-0052 (2). Sarcinomyces phaeomuriformis CBS 131.88 (2). Some 18S rRNA-genes were shown to habor two or three different introns (see the numbers in brackets) simultaneously.
A structural analysis of three of these introns (E. dermatitidis and Phaeoannellomyces elegans) indicated that they could be classified as group I introns. A distinctive feature of group I introns is their ability for autocatalytic splicing. For the two introns of Exophiala dermatitidis this autocatalytic splicing could be experimentally demonstrated using a novel non-radioactive assay (E-PCR). Phylogenetic analysis using 18s rRNA-gene sequences revealed that all these intron-haboring dematious hyphomycetes could be placed among species of the Herpotrichiellaceae.
Our-findings parallel observations of other groups that introns can occur in some nuclear 18S rRNA-genes of higher fungi as a consequence of their genetic mobility. The potential presence of such introns in fungal nuclear 18S rRNA-genes should be observed when applying techniques like ribotyping for (epidemic) identification of fungal pathogens.
Detailed Map of Mitochondrial Genom of Aspergillus carbonaris
Zsuzsanna Hamari, Ilona Pfeiffer, Betita Toth and Ferenc Kevei. Department of Microbiology, Attilai Jozsef University, H-6701 Szeged, P.O. Box 533, Hungary
Aspergillus carbonarius (Thom) is possibly the most distinct member of the black Aspergilli exhibiting some unique characters. Studying the molecular polymorphism of this species among thirteen examined strains two main mitochondrial DNA (mtDNA) groups could be distinguished (designated type 1 and type 2 respectively). These groups correspond to their nuclear rDNA types. The mtDNA type 1 could be divided into two subgroups (labelled type la and type 1b respectively) based on slight alterations of their RFLP patterns. Despite of the significant similarities of their restriction patterns considerable differences were estimated between the genome sizes of two subtypes (51-52 kb, 60-61 kb respectively). For interpretation of these differences between two subgroups an attempt was made to construct their more detailed restriction maps. Isolated mtDNA was digested by EcoRI and the fragments were separated by electrophoresis and cloned into pbluescript KS plasmid. The cloned fragments were digested by several restriction enzymes, such as EcoRV; Pstl; Hindlll; HincIl; Xhol; Kpnl; Xbal; BamHI;Haelll, Pvull. On the basis of these data linearized physical maps of both subtypes were developed. Digoxigenin-labelled hybridisation probes were applied for identifying the possible positions of certain regions.
Phylogenetic Analysis of the Genus Aspergillus Based on RFLP of Repetitive and Single Copy Sequences.
D.Hoffman-Zacharska1*, K. Spalik2, P.Stephien1. 1 Dept. of Genetics, University of Warsaw, 2 Dept. of Plant
Systematic and Geography , University of Warsaw, Warsaw, Poland current address - Dept. of Genetics, Inst. of Psychiatry and Neurology, Warsaw, Poland
Species of Aspergillus belong to the first fungal organisms that were cultivated on artificial media and studied for biochemical properties, they are also among the most common fungi in man's environment. Classical methods for systematic studies of this genus have been very successful and have provided a relatively good classification. However, morphological characters used for these studies probably do not reflect the real phylogenetic and evolutionary relationships between the taxa.
In our work to determine the relationship among several species of the genus (39 strains representing 5 subspecies and 12 sections) we used restriction fragment length polymorphism (RFLP) of the unit coding three classes of rRNA - 5.8S, 18S) 26S.
Using a plasmid containing the cloned rRNA unit from A.nidulans as a probe we found this method to be useful on the level of section. For more detailed analysis of strains representing section Nigri (22 strains) we additionally used RFLP analysis for a single copy gene - the gene coding glucoamylase from A.awamori.
Recombination of Mitochondrial DNA Following Directed Mitochondrial Transmission among Black Aspergilli
F. Kvei1, B. Toth1, A. Coenen2 Zs. Hamaril, J. Vargill, J.H, Croft3 1Department of Microbiology , Attila Jozsef University, H-6701 Szeged, P.O. Box 533, Hungary. 2Department of Genetics, Agricultural University, Wageningen, 6703 TIA, The Netherlands 3School of Biological Sciences, University of Birmingham, Birmingham, P.O.Box. 363, B15 2TT, U.K.
Strains of the Aspergillus niger aggregate can be divided to three main groups based on their HaellI-Bglll digested mtDNA patterns. The first and second groups correspond to A. niger and A. tubingensis species respectively, while the third type is represented only by some wild-type isolates derived from Brazil (mtDNA types 1, 2 and 3 correspond to rDNA groups I, II, and III, respectively). The mtDNA types I and 2 consist of several subgroups (labelled la - le, and 2a - 2f, respectively) [1]. Successful mitochondrial transfers were performed by protoplast fusion. Recombination of mtDNA occurred among strains representing different mtDNA groups or subgroups. All these strains showed full incompatibility in respect of nuclear complementation. Transfer experiments were carried out under selection pressure using a mitochondrial oligomycin resistant (oliR) mutant as a donor, which exhibits type la mtDNA and type I rDNA. Mitochondrial oliR progenies could be recovered following protoplast fusion in the presence of oligomycin by selecting for the nuclear phenotypes of the oliS recipient strains. The transfers were successful in all possible combinations. Within the group of strains belonging to mtDNA type I the transfers resulted in a single type of recombinant RFLP profile in each subgroup. The recombination events were more complex when the transfer of oliR occurred between strains representing different species. A great variety of recombined RFLP profiles of the partners appeared.
In mtDNA transfers la 2b and la 2d, resistant clones harbouring possibly unchanged donor mitochondria with the nuclear background of the recipient strain were also recovered in addition to the recombinant types.
1. Varga, J. et al. (1994) Can. J. Mcrobiol. 40: 612-621
Selection of mutS Homologs of Aspergillus nidulans
S. McLaughlin, A.B. Tomsett, H. Thijsl, H.W.J. van den Broekl and P. Strike. Department of Genetics and Microbiology, The University of Liverpool, Liverpool, England. L69 3BX. 1 Department of Genetics, Agricultural University Wageningin, The Netherlands.
Recent findings linking the mutS homolog (MSH2) to lymphoid and colon cancer in mice and humans respectively (Reitmair et al., 1995, and Fishel, et al., 1994), has renewed interest in the effects of mismatch repair genes in eukaryotic cells.
The bacterial mutS gene functions in the recognition and binding of a mismatched base pair (New et al., 1993). Saccharomyces cerevisiae has been found to encode 4 proteins (MSH 1 -4) which show strong amino acid similarity to mutS (Alani, et al., 1995).
The aim of this work is to isolate mutS homologs of Aspergillus nidulans by homology to genes from related species including Neurospora crassa and S. cerevisiae and to investigate the effect of knockout mutations on the polarity of gene conversion found to exist between the niiA and niaD genes of Aspergillus nidulans (Thijs, et al., 1995).
Considerable sequence homology (up to 78%) exists in the 3' regions of the mutS genes of various bacterial and eukaryotic species. Selection of mutS homologs of A. nidulans was based on the construction of degenerate primers from these regions of high homology. Primers were used with both A. nidulans genomic DNA and a cDNA library. Selected PCR products were partially sequenced and used as probes against a cDNA library for selection of a complete copy of the mutS homologs.
Genomic Comparison among Wild-type and Mutant Strains of Phaffia rhodozyma
Agnes Nagy1, Zsuzsanna Palagyi, Csaba Vagvolgyi and Lajos Ferenczy. 1 Chemical Works of Gedeon Richter Ltd., P.O. Box 27, H-1475, Budapest, Hungary. Department of Microbiology, Attila Jozsef Universi ty, P. 0. Box 533, H6701, Szeged, Hungary
P. rhodozyma is the onlv known yeast that produces the pigment astaxanthin (3,3'-dihydroxy- , -carotene-4,4'-dione) as its principal carotenoid. Though this can be manufactured svnthetically, the demand for the available natural sources has substantially increased.
Eighty mutants obtained from two P. rhodozyma strains (ATCC 24203 and ATCC 24229) by a recently described method (1) were investigated. Protoplast fusion experiments were carried out between mutants with auxotrophic and morphologic (colour) markers and the resulting fusion products were isolated. CHEF separation of the chromosomal size DNAs (2) revealed several new chromosomal patterns after mutagen treatment. Analysis of chromosomal rearrangements proved useful for a more exact estimation of chromosome number and genome size. No correlation was found between a given type of chromosomal aberration and a phenotypic character. When hybrid strains resulting from protoplast fusion were analysed- both the disappearance of certain bands and the appearance of new bands were detected.
(1)Zs.Palagyi, A. Nagy, Cs. Vagvolgyi and L. Ferenczy (1995) Biotechnol. Tech. 9, 401-402.
(2)A. Nagy, N. Garamszegi, Cs. Vagvolgyi and L. Ferenczy (1994) FEMS Microbiol. Lett. 123, 315-318.
Genetic Inactivation of Mitochondrial Acyl Carrier Protein in N. crassa and S. cerevisiae
Regina Schneider, Benedikt Brors, Christoph Krull, Michael Massow, and Hanns Weiss. Institut fur Biochemie, Heinrich-Heine-Universitat Dusseldorf, UniversitatsstraBe 1, 40225 Dusseldorf, Federal Republic of Germany
The nuclear genes encoding the mitochondrial acyl carrier protein (acp-1, ACPI) were disrupted in Neurospora crassa and Saccharomyces cerevisiae. In N. crassa, the acp-I is a peripheral subunit of the respiratory NADH:ubiquinone oxidoreductase (complex 1). S. cerevisiae lacks complex I and its ACPI appears to be located in the mitochondrial matrix. The loss of the acp- I in N. crassa causes two biochemical lesions. First, the peripheral part of complex I is not assembled, and the assembly of the membrane part is disturbed. The respiratory ubiquinol:cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV), however, are made in normal amounts. Second, the lysophospholipid content of mitochondrial membranes is increased fourfold. In S. cerevisiae, the loss of the ACP I leads to a pleiotropic respiratory-deficient phenotype
The phenotype of the acp-1 deficient mutant of N. crassa was restored by complementation with the acp-1 gene of Aspergillus niger. This was accomplished by the use of the heterologous selection marker bar providing resistance against the herbicide BASTAreg. (phosphinotricin).
Studies on the Function and the Regulation of the AOX Gene from Penicillium chrysogenum
Edith Schreiner, Klaus Holzmann and Helmut Schwab. Institut fur Biotechnologie, Arbeitsgruppe Genetik, TU Graz
A-8010 Graz, Austria
The specifically regulated aox gene, dependent on the physiological state and on external pH conditions shows strong homology to alcohol oxidases of methylotrophic yeasts.
In order to investigate the functionality and the regulation of the aox promoter we fused a 0.8kb promoter fragment to the E.coli glucuronidase gene (uidA) as the reporter gene. A transformant containing one single copy integrated at the homologous marker gene was used for further studies. -glucuronidase expression was examined on plates under conditions of different carbon sources adjusted at different pH values.
To analyse the function of the aox gene we overexpressed this gene in E.coli as well as in P. chrysogenum. On the other hand we constructed plasmids to inactivate the aox gene by targeted gene disruption. We are currently analysing the respective transformants.
Using Monospore Pellets in Genetic and Population Analysis of Pleurotus ostratus
I. Druzhinina, A. Shnyreva, I. Insarova and Y. T.Dyakov. Department of Mycology and Algology, Moscow Lomonosov University, Moscow 119899, Russia
We report here the novel method of producing monospore mycelial pellets from the spore prints of Pleurotus ostreatus fruit bodies which is less time consuming and useful for genetic analysis. The culturing conditions under which each basidiospore gives rise to an individual haploid pellet are described. A 20-30 offspring pellets can be obtained from one basidiome (dikaryotic parent) in a flask. The resultant monospore progeny can then be tested for genetic polymorphism(s) using isozyme protein electrophoresis and RAPD technique, a mycelium quantity of each pellet (a basidiospore derived monokaryon ) being enough for the analysis.
P. ostreatus presents an exellent model organism because of well-established haplo-diploid life cycle and the following advantages:
- dikaryotic and homokaryotic clones can be reproduced on artificial media perrmtting analysis of their mating relationships; - mating criteria are easily testable in Pleurotus;
- along with multiallelic mating compatibility system the fungus has genetically controled somatic incompatibility system which delimits individual clones.
Spore prints of different origins are presently being screened. Data on clonal and population structure, equation between outbreeding and inbreeding processes in P. ostreatus is expected to be obtained.
The results will be discussed in relation to the population ecology and reproductive strategies in Pleurotus.
Mitochondrial DNA Recombination among Compatible Strains of the Aspergillus niger Aggregate Without Using Selection Pressure
Beata Toth, Zsuzsanna Hamari and Ferenc Kevei. Department of Microbiology, Attila Jozsef University H-6701, Szeged,
P.O. Box 533, Hungary
Heterokaryon formation is a rare event between individual isolates of asexual black Aspergillus species (section Nigri), even if they derived from the same geographical area. The reason for this extreme heterokaryon incompatibility is still not understood. Heterokaryosis might occur very rarely only between those strains which harbour the same molecular markers, but never occur between isolates bearing polymorphic molecular characters, even if they represent the same species. The lack of genetic or molecular markers also hampered the examination of mtDNA inheritance even between compatible strains. When oligomycin resistant mitochondria were transmitted into incompatible sensitive partners (la 2b and la 2d), two resistant clones were isolated bearing possibly unchanged donor mitochondria as regards their mtDNA RFLP patterns (mtDNA type la) with the nuclear background of the oliS recipient partner (rDNA type II, and other phenotypic characters). The resistant strains harbouring either recombinant or substituted mtDNAs were crossed with a compatible sensitive parent of isogenic origin by anastomosis. Oligomycin was not applied for selection during heterokaryon formation. Heterokaryons possessing mixed mitochondrial populations showed different growth rates. After prolonged cultivation, conidia of the heterokaryons were harvested. Both parental nucleotypes could be recovered from the progeny. An overwhelming majority of the progeny harbours recombined mitochondria. This result suggests that mitochondrial recombination may occur without any selection pressure. This model is a useful tool for studying mitochondrial inheritance and the mechanism of mtDNA recombination following parasexual processes. These types of recombined mtDNA RFLP profiles will be compared with those isolated following strong selection procedure.
Mitochondrial DNA Inheritance in Aspergillus nidulans-A. tertazonus Hybrids
Janos Varga1, Kristztina Kesztyus1 and James H. Croft2. 1 Department of Microbiology, Attila Jozsef University,
PO Box 533, H-6701 Szeged, Hungary. 2 School of Biological Sciences, University of Birmingham,
Brmingham, PO Box 363, B15 2TT UK
Interspecific hybrids were produced between an Aspergillus nidulans ‘master strain', and an A. tertazonus auxotrophic mutant by protoplast fusion. The haploid segregants obtained after treating the allodiploid hybrids with haploidizing agents like benomyl were examined for their niitochondrial DNA (mtDNA) restriction patterns. Without selecting for any of the parental mtDNA genomes, some of the progeny harboured recombinant mtDNAs, while some others carried the mtDNA of the A. nidulans parent. The observed recombinant mtDNAs were different from those obtained by selecting for the mitochondrial oligomycin resistance marker of the A. tertazonus parent. Only the mtDNA patterns of the A. nidulans parent were observed in the progeny when selection was carried out for the mitochondrial oligomycin resistance marker of A. nidulans. Simple physical maps of the two different recombinant mtDNAs were constructed and compared to the parental mitochondrial genomes. The recombinant mtDNAs differed from the parental mtDNAs in the presence of a new HaeIII fragment, which indicates that the recombination event took place in the coxl gene. The two recombinant mtDNAs possibly differ from each other in the presence or absence of an intronic sequence in the cog gene.
Studies on the Electron-pathway of Neurospora crassa Complex I Using Site-directed Mutagenesis
Ute Wobschall, Christoph Krull, Hanns Weiss. Institut fur Biochemie, Heinrich-Heine-Universitat Dusseldorf, Universitatsstr. 1, 40225 Dusseldorf, Germany
The NADH:ubiquinone oxidoreductase (complex 1) is the most complicated enzyme of the respiratory chain. The eucaryotic complex I couples the transfer of two electrons from NADH to ubiquinone with the translocation of four protons across the inner mitochondrial membrane. In the ascomycete Neurospora crassa complex I consists of some 35 subunits with one FMN and four EPR detectable iron-sulphur clusters serving as prosthetic groups.
The 78 kDa subunit possesses two binding motifs for FeS clusters. For further understanding of the electron-pathway we inactivated the gene NUO 78 by homologous recombination with an in vitro deleted copy of the gene. We used the heterologous hph-gene for a dominant selection marker.
Moreover, we use site-directed mutagenesis for specific analysis of FeS clusters by exchanging distinct amino acids of putative binding motifs. Using the bar-gene, which confers resistance to the herbicide Basta, as another heterologous dominant marker, the deletion mutant nuo 78 can be transformed with the point-mutated copy of the gene NUO 78. FeS clusters with changed characteristics can subsequently be identified by EPR spectroscopy.
Detection of Endoparasitic Fungi in Nematodes
Yvonne Persson, Agnes Roumegooux, Susanner Erland and Hans-Borje Jansson. Dept of Microbial Ecology, Ecology Building, Lund University 223 62 Lund, Sweden
Many nematode species are parasites of plants and animale, including economically important crops and livestock. Nematodes, in their turn, can be parasitised by several parasitic fungi, the so-called nematophagous fungi. These fungi are used for development of control methods for nematode pests in plants and animals.
Endoparasitic, nematophagous fungi develop their hyphal system within thier host where they spend their entire vegetative lives; only spore bearing structures will be produced outside the body of the nematodes.
The purpose of this work was to investigate the possibility to use molecular techniques to detect and identify endoparasitic fungi inside single infected nematodes. PCR was used to amplify the ITS (internal transcribed spacer) region of the rDNA. The amplification product was cut with different restriction enzymes to produce RFLP patterns.
Six different species of endoparasitic fungi, that were possible to identify in axenic culture by the PCR/RFLP method were used to infect nematodes. The amplification products from the infected nematodes were represented by two bands: one band resulted from the amplification of the nematode ITS-region and the other band from the ITS of the fungi. After restriction analysis it was possible to identify three out of six fungal species, within the infected host.
Ectomycorrhizal Community Diversity in Forests with Different Nitrogen Deposition, Measured by PCR/RFLPS from Single Mycorrhizas
Susanne Erland, Shahid Mahmood, Tina Hohsson and Roger Finlay, Dept of Microbial Ecology, ecology Building, Lund University, 223 62 Lund Swede
Circumstantial evidence suggests that functional progerties of ectomycorrhiazal communities can be influenced by antrhropogenic stress. To be able to study how, we need information about changes in themycorrhizal community at the level of individual mycorrhizal roots, since those are the organs for nutrient exchange. Ideally such studies should be followed by directed physiological laboratory studies on the species identified to be of interest. We have used PCR/RFLP methods to analyse the ITS-region of the ribosomal DNA of the mycobionts forming mycorrhizas on spruce at two sites with different nitrogen deposition. The data revealed a total of sixeen different ITS types in both forests, and three of thosewere found at both sites. In the high N-deposition site a total of eight ITS-types were found wheras the low N-deposition site had fifteen different ITS types. Identification of the ITS- types by comparison with those of identified fruitbodies from the same regrion is in progress. Our data show a higher ectomycorrhizal diversity in the non-polluted site. Inter and intra specific variation in the ability to utilise organic-N sources is now being examined in the mycobionts from both sites to determine whether changes in community structure can be explained in terms of differences in nitrogen deposition.
Development of an Easy and Rapid Method to Screen Transformants Directly by PCR
Cora van Zeijl and Peter Punt
TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, PO Box 5815, 2280 HV Rijswijk, The Netherlands
A method has been developed for screening large numbers of transformants of different filamentous fungi by PCR. Colonies were grown in 96 well microtiter plates. Protoplasts were generated in these wells by NOVOZYM234 treatment and thewe were heated to 95 C. The resulting solution sould be immediately be used for PCR. Different PCR enzymes were tested for their performance on this template. PCR fragments sized up to 6 kb could be reproducibly detected,
In an application Beauveria bassiana transformants were screened for a deletion in a gene docing for a cytochrome P450 enzyme, for which no phenotype was available. In a screen of 250 colonies 5 transformants with a deletion genotypoe were detected using this method.
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Last modified 8/13/96 KMC